Poly(ADP-ribose) polymerase (PARP, also called poly(ADP-ribose) synthetase, or PARS) is a nuclear enzyme which catalyzes the synthesis of poly(ADP-ribose) chains from NAD+ in response to single-stranded DNA breaks as part of the DNA repair process (de Murcia et al. Trends Biochem. Sci. 1994, 19, 172; Alvarez-Gonzalez et al. Mol. Cell. Biochem. 1994, 138, 33). The chromatin-associated protein substrates for ADP-ribosylation, which include histones, DNA metabolizing enzymes and PARP itself, are modified on surface glutamate residues. PARP catalyzes attachment of one ADP-ribose unit to the protein (initiation), followed by polymerization of as many as 200 ADP-ribose monomers (elongation) via 2′-1″ glycosidic linkages. In addition, PARP catalyzes branching of the polymer at a lower frequency.
The role of PARP in the DNA repair process is incompletely defined. The binding of PARP to nicked double-stranded DNA is suggested to facilitate the repair process by transiently blocking DNA replication or recombination. The subsequent poly(ADP-ribosyl)ation of PARP and histones may result in introduction of a substantial negative charge, causing repulsion of the modified proteins from the DNA. The chromatin structure is then proposed to relax, enhancing the access of DNA repair enzymes to the site of damage.
Excessive activation of PARP in response to cell damage or stress is hypothesized to result in cell death (Sims et al. Biochemistry 1983, 22, 5188; Yamamoto et al. Nature 1981, 294, 284). Activation of PARP by DNA strand breaks may be mediated by nitric oxide (NO) or various reactive oxygen intermediates. When the degree of DNA damage is large, PARP may catalyze a massive amount of poly(ADP-ribosyl)ation, depleting the cell's levels of NAD+. As the cell attempts to maintain homeostasis by resynthesizing NAD+, levels of ATP may decrease precipitously (since synthesis of one molecule of NAD+ requires four molecules of ATP) and the cell may die through depletion of its energy stores.
Activation of PARP has been reported to play a role in cell death in a number of disease states, suggesting that PARP inhibitors would have therapeutic efficacy in those conditions. Enhanced poly(ADP-ribosyl)ation has been observed following focal cerebral ischemia in the rat, consistent with activation of PARP in stroke (Tokime et al. J. Cereb. Blood Flow Metab. 1998, 18, 991). A substantial body of published pharmacological and genetic data supports the hypothesis that PARP inhibitors would be neuroprotective following cerebral ischemia, or stroke. Inhibitors of PARP protected against NMDA- or NO-induced neurotoxicity in rat cerebral cortical cultures (Zhang et al., Science 1994, 263, 687; Eliasson et al. Nature Med. 1997, 3, 1089). The degree of neuroprotection observed for the series of compounds directly paralleled their activity as PARP inhibitors.
Inhibitors of PARP may also display neuroprotective efficacy in animal models of stroke. The potent PARP inhibitor DPQ (3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone) (Suto et al., U.S. Pat. No. 5,177,075) provided a 54% reduction in infarct volume in a rat model of focal cerebral ischemia (permanent MCAo and 90 min bilateral occlusion of the common carotid artery) following i.p. dosing (10 mg/kg) two hours prior to and two hours after the initiation of ischemia (Takahashi et al. Brain Res. 1997, 829, 46). Intracerebroventricular administration of a less potent PARP inhibitor, 3-aminobenzamide (3-AB), yielded a 47% decrease in infarct volume in mice following a two hour occlusion of the MCA by the suture thread method (Endres et al. J. Cereb. Blood Flow Metab. 1997, 17, 1143). Treatment with 3-AB also enhanced functional recovery 24 hours after ischemia, attenuated the decrease in NAD+ levels in ischemic tissues, and decreased the synthesis of poly(ADP-ribose) polymers as determined by immunohistochemistry. Similarly, 3-AB (10 mg/kg) significantly reduced infarct volume in a suture occlusion model of focal ischemia in the rat (Lo et al. Stroke 1998, 29, 830). The neuroprotective effect of 3-AB (3-30 mg/kg, i.c.v.) was also observed in a permanent middle cerebral artery occlusion model of ischemia in the rat (Tokime et al. J. Cereb. Blood Flow Metab. 1998, 18, 991).
The availability of mice in which the PARP gene has been rendered non-functional (Wang, Genes Dev. 1995, 9, 509) has also helped to validate the role of PARP in neurodegeneration. Neurotoxicity due to NMDA, NO, or oxygen-glucose deprivation was virtually abolished in primary cerebral cortical cultures from PARP−/− mice (Eliasson et al. Nature Med. 1997, 3, 1089). In the mouse suture thread model of ischemia, an 80% reduction in infarct volume was observed in PARP−/− mice, and a 65% reduction was noted in PARP+/− mice. In Endres et al. (1997), there was reported a 35% reduction in infarct volume in PARP−/− mice and a 31% reduction in PARP+/− animals. In addition to neuroprotection, PARP−/− mice demonstrated an improvement in neurological score and displayed increased NAD+ levels following ischemia.
Preclinical evidence also exists which suggests that PARP inhibitors may be efficacious in the treatment of Parkinson's disease. This is because loss of dopaminergic neurons in the substantia nigra is a hallmark of Parkinson's disease. Treatment of experimental animals or humans with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) replicates the loss of dopaminergic neurons and the motor symptoms of Parkinson's disease. MPTP activates PARP in the substantia nigra, and mice lacking PARP are resistant to the neurodegenerative effects of MPTP (Mandir et al. Proc. Nat. Acad. Sci. 1999, 96, 5774). Similarly, the PARP inhibitor 3-aminobenzamide is reported to attenuate the loss of NAD+ in the striatum following administration of MPTP to mice (Cosi et al. Brain Res. 1998, 809, 58).
Activation of PARP has been implicated in the functional deficits that may result from traumatic brain injury and spinal cord injury. In a controlled cortical impact model of traumatic brain injury, PARP−/− mice displayed significantly improved motor and cognitive function as compared to PARP+/+ mice (Whalen et al. J. Cereb. Blood Flow Metab. 1999, 19, 835). Peroxynitrite production and PARP activation have also been demonstrated in spinal cord-injured rats (Scott et al. Ann. Neurol. 1999, 45, 120). These results suggest that inhibitors of PARP may provide protection from loss of function following head or spinal trauma.
The role of PARP as a mediator of cell death following ischemia and reperfusion may not be limited to the nervous system. In this connection, a recent publication reported that a variety of structurally distinct PARP inhibitors, including 3-AB and related compounds, reduce infarct size following cardiac ischemia and reperfusion in the rabbit (Thiemermann et al. Proc. Nat. Acad. Sci. 1997, 94, 679). In the isolated perfused rabbit heart model, inhibition of PARP reduced infarct volume and contractile dysfunction following global ischemia and reperfusion. Skeletal muscle necrosis following ischemia and reperfusion was also attenuated by PARP inhibitors. Similar cardioprotective effects of 3-AB in a rat myocardial ischemia/reperfusion model were reported by Zingarelli and co-workers (Zingarelli et al. Cardiovascular Research 1997, 36, 205). These in vivo results are further supported by data from experiments in cultured rat cardiac myocytes (Gilad et al. J. Mol. Cell. Cardiol. 1997, 29, 2585). Inhibitors of PARP (3-AB and nicotinamide) protected the myocytes from the reductions in mitochondrial respiration observed following treatment with oxidants such as hydrogen peroxide, peroxynitrite, or nitric oxide donors. The genetic disruption of PARP in mice was recently demonstrated to provide protection delayed cellular injury and production of inflammatory mediators following myocardial ischemia and reperfusion (Yang et al. Shock 2000, 13, 60). These data support the hypothesis that administration of a PARP inhibitor could contribute to a positive outcome following myocardial infarction. A particularly useful application of a PARP inhibitor might involve administration concurrent with a treatment designed to reperfuse the affected area of the heart, including angioplasty or a clot-dissolving drug such as tPA.
The activity of PARP is also implicated in the cellular damage that occurs in a variety of inflammatory diseases. Activation of macrophages by pro-inflammatory stimuli may result in the production of nitric oxide and superoxide anion, which combine to generate peroxynitrite, resulting in formation of DNA single-strand breaks and activation of PARP. The role of PARP as a mediator of inflammatory disease is supported by experiments employing PARP−/− mice or inhibitors of PARP in a number of animal models. For example, joints of mice subjected to collagen-induced arthritis contain nitrotyrosine, consistent with generation of peroxynitrite (Szabo et al. J. Clin. Invest. 1998, 100, 723). The PARP inhibitor 5-iodo-6-amino-1,2-benzopyrone reduced the incidence and severity of arthritis in these animals, decreasing the severity of necrosis and hyperplasia of the synovium as indicated by histological examination. In the carrageenan-induced pleurisy model of acute local inflammation, 3-AB inhibited the histological injury, pleural exudate formation and mononuclear cell infiltration characteristic of the inflammatory process (Cuzzocrea et al. Eur. J. Pharmacology 1998, 342, 67).
Results from rodent models of colitis suggest that PARP activation may be involved in the pathogenesis of inflammatory bowel disease (Zingarelli et al. Gastroenterology 1999, 116, 335). Administration of trinitrobenzene sulfonic acid into the lumen of the bowel causes mucosal erosion, neutrophil infiltration, and the appearance of nitrotyrosine. Deletion of the PARP gene or inhibition of PARP by 3-AB decreased tissue damage and attenuated neutrophil infiltration and nitrotyrosine formation, suggesting that PARP inhibitors may be useful in the treatment of inflammatory bowel disease.
A role for PARP in the pathogenesis of endothelial dysfunction in models of endotoxic shock has also been proposed (Szabo et al. J. Clin. Invest. 1997, 100, 723). This is because PARP inhibition or genetic deletion of PARP may protect against the decrease in mitochondrial respiration that occurs following treatment of endothelial cells with peroxynitite.
The activation of PARP is involved in the induction of experimental diabetes initiated by the selective beta cell toxin streptozocin (SZ). Substantial breakage of DNA may be induced by SZ, resulting in the activation of PARP and depletion of the cell's energy stores as described above in Yamamoto et al. (1981). In cells derived from PARP−/− mice, exposure to reactive oxygen intermediates results in attenuated depletion of NAD+ and enhanced cell viability relative to wild-type cells (Heller et al. J. Biol. Chem. 1995, 270, 11176). Similar effects were observed in wild-type cells treated with 3-AB. Subsequent studies in mice treated with SZ indicated that deletion of the PARP gene provides protection against loss of beta cells (Burkart et al. Nature Med. 1999, 5, 314; Pieper et al. Proc. Nat. Acad. Sci. 1999, 96, 3059). These observations support the hypothesis that an inhibitor of PARP may have therapeutic utility in the treatment of type I diabetes.
Another potential therapeutic utility of PARP inhibitors involves enhancement of the anti-tumor activity of radiation or DNA-damaging chemotherapeutic agents (Griffin et al. Biochemie 1995, 77, 408). Since polyADP-ribosylation occurs in response to these treatments and is part of the DNA repair process, a PARP inhibitor might be expected to provide a synergistic effect.
Like PARP, protein kinases play a critical role in the control of cells. In particular, kinases are known to be involved in cell growth and differentiation. Aberrant expression or mutations in protein kinases have been shown to lead to uncontrolled cell proliferation, such as malignant tumor growth, and various defects in developmental processes, including cell migration and invasion, and angiogenesis. Protein kinases are therefore critical to the control, regulation, and modulation of cell proliferation in diseases and disorders associated with abnormal cell proliferation. Protein kinases have also been implicated as targets in central nervous system disorders such as Alzheimer's disease, inflammatory disorders such as psoriasis, bone diseases such as osteoporosis, atherosclerosis, restenosis, thrombosis, metabolic disorders such as diabetes, and infectious diseases such as viral and fungal infections.One of the most commonly studied pathways involving kinase regulation is cellular signaling from receptors at the cell surface to the nucleus. Generally, the pattern of expression, ligand availability, and the array of downstream signal transduction pathways that are activated by a particular receptor, determine the function of each receptor. One example of a pathway includes a cascade of kinases in which members of the growth factor receptor tyrosine kinases deliver signals via phosphorylation to other kinases such as Src tyrosine kinase, and the Raf, Mek and Erk serine/threonine kinase families. Each of these kinases is represented by several family members that play related but functionally distinct roles. The loss of regulation of the growth factor signaling pathway is a frequent occurrence in cancer as well as other disease states (Fearon, Genetic Lesions in Human Cancer, Molecular Oncology 1996, 143-178).
One receptor tyrosine kinase signaling pathway includes the vascular endothelial growth factor (VEGF) receptor kinase. It has been shown that binding of VEGF to the receptor VEGFR2 affects cell proliferation. For instance, binding of VEGF to the VEGFR-2/flt-1 receptor, which is expressed primarily on endothelial cells, results in receptor dimerization and initiation of a complex cascade which results in growth of new blood vessels (Korpelainen and Alitalo, Curr. Opin. Cell. Biol. 1998, 10, 159). Suppression of formation of new blood vessels by inhibition of the VEGFR tyrosine kinases would have utility in a variety of diseases, including treatment of solid tumors, diabetic retinopathy and other intraocular neovascular syndromes, macular degeneration, rheumatoid arthritis, psoriasis, and endometriosis.
An additional kinase signal transduction is the stress-activated protein kinase (SAPK) pathway (Ip and Davis Curr. Opin. Cell Biol. 1998, 10, 205). In response to stimuli such as cytokines, osmotic shock, heat shock, or other environmental stress, the pathway is activated and dual phosphorylation of Thr and Tyr residues within a Thr-Pro-Tyr motif of the c-jun N-terminal kinases (JNKs) is observed. Phosphorylation activates the JNKs for subsequent phosphorylation and activation of various transcription factors, including c-Jun, ATF2 and ELK-1.
The JNKs are mitogen-activated protein kinases (MAPKs) that are encoded by three distinct genes, jnk1, jnk2 and jnk3, which can be alternatively spliced to yield a variety of different JNK isoforms (Gupta et al., EMBO J. 1996, 15, 2760). The isoforms differ in their ability to interact with and phosphorylate their target substrates. Activation of JNK is performed by two MAPK kinases (MAPKK), MKK4 and MKK7. MKK4 is an activator of JNK as well as an additional MAPK, p38, while MKK7 is a selective activator of JNK. A number of MAPKK kinases are responsible for activation of MKK4 and MKK7, including the MEKK family and the mixed lineage kinase, or MLK family. The MLK family is comprised of six members, including MLK1, MLK2, MLK3, MLK6, dual leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK). MLK2 is also known as MST (Katoh, et al. Oncogene, 1994, 10, 1447). Multiple kinases are proposed to be upstream of the MAPKKKs, including but not restricted to germinal center kinase (GCK), hematopoietic progenitor kinase (HPK), and Rac/cdc42. Specificity within the pathway is contributed, at least in part, by scaffolding proteins that bind selected members of the cascade. For example the JNK interacting protein-1 (JIP-1) binds HPK1, DLK or MLK3, MKK7 and JNK, resulting in a module which enhances JNK activation (Dickens et al. Science 1997, 277, 693).
Manipulation of the activity of the SAPK pathway can have a wide range of effects, including promotion of both cell death and cell survival in response to various pro-apoptotic stimuli. For example, down-regulation of the pathway by genetic disruption of the gene encoding JNK3 in the mouse provided protection against kainic acid-induced seizures and prevented apoptosis of hippocampal neurons (Yang et al. Nature 1997, 389, 865). Similarly, inhibitors of the JNK pathway such as JIP-1 inhibit apoptosis (Dickens, supra). In contrast, the activity of the JNK pathway appears to be protective in some instances. Thymocytes in which MKK4 has been deleted display increased sensitivity to CD95- and CD3 mediated apoptosis (Nishina et al. Nature 1997, 385, 350). Overexpression of MLK3 leads to transformation of NIH 3T3 fibroblasts (Hartkamp et al. Cancer Res. 1999, 59, 2195).
An area the present invention is directed toward is identification of compounds that modulate the MLK members of the SAPK pathway and promote either cell death or cell survival Inhibitors of MLK family members would be anticipated to lead to cell survival and demonstrate therapeutic activity in a variety of diseases, including chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease and acute neurological conditions such as cerebral ischemia, traumatic brain injury and spinal injury. Inhibitors of MLK members leading to inhibition of the SAPK pathway (JNK activity) would also display activity in inflammatory diseases and cancer.
An additional member of the MAP kinase family of proteins is the p38 kinase. Activation of this kinase has been implicated in the production of proinflammatory cytokines such as IL-1 and TNF. Inhibition of this kinase could therefore offer a treatment for disease states in which disregulated cytokine production is involved.
The signals mediated by kinases have also been shown to control cell growth, cell death and differentiation in the cell by regulating the processes of the cell cycle. A family of kinases called cyclin dependent kinases (CDKs) controls progression through the eukaryotic cell cycle. The loss of control of CDK regulation is a frequent event in hyperproliferative diseases and cancer.
Inhibitors of kinases involved in mediating or maintaining particular disease states represent novel therapies for these disorders. Examples of such kinases include Src, raf, the cyclin-dependent kinases (CDK) 1, 2, and 4 and the checkpoint kinases Chk1 and Cds1 in cancer, CDK2 or PDGF-R kinase in restenosis, CDK5 and GSK3 kinases in Alzheimer's Disease, c-Src kinase in osteoporosis, GSK3 kinase in type-2 diabetes, p38 kinase in inflammation, VEGFR 1-3 and TIE-1 and -2 kinases in angiogenesis, UL97 kinase in viral infections, CSF-1R kinase in bone and hematopoietic diseases, and Lck kinase in autoimmune diseases and transplant rejection.
A variety of compounds which are described as PARP or kinase inhibitors have been reported in the literature including Banasik et al. J. Biol. Chem. 1992, 267, 1569 and Banasik et al. Mol. Cell. Biochem. 1994, 138, 185. Many other PARP inhibiting compounds have been the subject of patents. For example, compounds that are described as PARP inhibitors are disclosed in WO 99/08680, WO 99/11622, WO 99/11623, WO 99/11624, WO 99/11628, WO 99/11644, WO 99/11645, WO 99/11649, WO 99/59973, WO 99/59975 and U.S. Pat. No. 5,587,384.
Structurally related compounds, which are described as having activities other than PARP inhibition, are disclosed in WO 99/47522, EP 0695755, and WO 96/28447. Other structurally related compounds, their syntheses and precursors are disclosed in Piers et al. J. Org. Chem. 2000, 65, 530, Berlinck et al. J. Org. Chem. 1998, 63, 9850, McCort et al. Tetrahedron Lett. 1999, 40, 6211, Mahboobi et al. Tetrahedron 1996, 52, 6363, Rewcastle et al. J. Med. Chem. 1996, 39, 918, Harris et al. Tetrahedron Lett. 1993, 34, 8361, Moody et al. J. Org. Chem. 1992, 57, 2105, Ohno et al. Heterocycles 1991, 32, 1199, Eitel et al. J. Org. Chem. 1990, 55, 5368, Kruto{hacek over (s)}íková et al. Coll. Czech. Chem. Commun. 1988, 53, 1770, Muchowski et al. Tetrahedron Lett. 1987, 28, 3453, Jones et al. J. Chem. Soc., Perkin Trans. I 1984, 2541, Noland et al. J. Org. Chem. 1983, 48, 2488, Jones et al. J. Org. Chem. 1980, 45, 4515, Leonard et al. J. Am. Chem. Soc. 1976, 98, 3987, Rashidan et al. Arm. Khim. Zh. 1968, 21, 793, Abrash et al. Biochemistry 1965, 4, 99, U.S. Pat. No. 5,728,709, U.S. Pat. No. 4,912,107, EP 0768311, JP 04230385, WO 99/65911, WO 99/41276, WO 98/09967, and WO 96/11933.
Because of the potential role in therapeutically treating neurodegenerative disorders, cancers, and other PARP and kinase related diseases, PARP and kinase inhibitors are an important class of compounds requiring further discovery, exploration, and development. Although, a wide variety of PARP and kinase inhibitors are known, many suffer from problems such as toxicity, poor solubility, and limited efficacy, which prevent practical therapeutic use and preclude further development into effective drugs. Thus, there is a current and immediate need for new PARP and kinase inhibitors for the treatment of PARP and kinase related diseases. The present invention is directed to this, as well as other important ends.